Table_1_T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice.DOCX

Primary familial brain calcification (PFBC) is a rare neurodegenerative and neuropsychiatric disorder characterized by bilateral symmetric intracranial calcification along the microvessels or inside neuronal cells in the basal ganglia, thalamus, and cerebellum. Slc20a2 homozygous (HO) knockout mice are the most commonly used model to simulate the brain calcification phenotype observed in human patients. However, the cellular and molecular mechanisms related to brain calcification, particularly at the early stage much prior to the emergence of brain calcification, remain largely unknown. In this study, we quantified the central nervous system (CNS)-infiltrating T-cells of different age groups of Slc20a2-HO and matched wild type mice and found CD45+CD3+ T-cells to be significantly increased in the brain parenchyma, even in the pre-calcification stage of 1-month-old -HO mice. The accumulation of the CD3+ T-cells appeared to be associated with the severity of brain calcification. Further immunophenotyping revealed that the two main subtypes that had increased in the brain were CD3+ CD4− CD8– and CD3+ CD4+ T-cells. The expression of endothelial cell (EC) adhesion molecules increased, while that of tight and adherents junction proteins decreased, providing the molecular precondition for T-cell recruitment to ECs and paracellular migration into the brain. The fusion of lymphocytes and EC membranes and transcellular migration of CD3-related gold particles were captured, suggesting enhancement of transcytosis in the brain ECs. Exogenous fluorescent tracers and endogenous IgG and albumin leakage also revealed an impairment of transcellular pathway in the ECs. FTY720 significantly alleviated brain calcification, probably by reducing T-cell infiltration, modulating neuroinflammation and ossification process, and enhancing the autophagy and phagocytosis of CNS-resident immune cells. This study clearly demonstrated CNS-infiltrating T-cells to be associated with the progression of brain calcification. Impairment of blood–brain barrier (BBB) permeability, which was closely related to T-cell invasion into the CNS, could be explained by the BBB alterations of an increase in the paracellular and transcellular pathways of brain ECs. FTY720 was found to be a potential drug to protect patients from PFBC-related lesions in the future.原发性家族性脑钙化（PFBC，Primary familial brain calcification）是一种罕见的神经退行性及神经精神疾病，以双侧对称性颅内钙化为特征，钙化沉积于基底神经节、丘脑和小脑的微血管壁内或神经元细胞内部。Slc20a2纯合（HO，homozygous）敲除小鼠是模拟人类患者脑钙化表型最常用的动物模型。然而，与脑钙化相关的细胞及分子机制，尤其是在脑钙化出现前的早期阶段，目前仍未完全阐明。本研究对不同年龄组的Slc20a2纯合敲除小鼠及匹配的野生型小鼠的中枢神经系统（CNS，central nervous system）浸润T细胞进行了定量分析，发现即使在1月龄纯合敲除小鼠的钙化前阶段，其脑实质内的CD45+CD3+ T细胞数量也显著升高。CD3+ T细胞的聚集似乎与脑钙化的严重程度相关。进一步的免疫表型分析显示，脑内数量增多的两大主要T细胞亚型为CD3+CD4−CD8− T细胞和CD3+CD4+ T细胞。内皮细胞（EC，endothelial cell）的黏附分子表达上调，而紧密连接蛋白及黏着连接蛋白的表达下调，这为T细胞黏附内皮细胞并通过旁细胞迁移进入脑实质提供了分子前提。研究人员观察到淋巴细胞与内皮细胞膜的融合以及CD3相关金颗粒的跨细胞迁移，这表明脑内皮细胞的转胞吞作用增强。外源性荧光示踪剂及内源性IgG和白蛋白渗漏实验也证实了脑内皮细胞转细胞通路的受损。芬戈莫德（FTY720）可显著减轻脑钙化，其机制可能为减少T细胞浸润、调节神经炎症及骨化过程，并增强中枢神经系统驻留免疫细胞的自噬与吞噬作用。本研究明确证实中枢神经系统浸润T细胞与脑钙化的进展密切相关。血脑屏障（BBB，blood–brain barrier）通透性受损与T细胞向中枢神经系统侵袭紧密相关，其机制可归因于脑内皮细胞旁细胞及转细胞通路的异常改变。芬戈莫德有望成为未来预防PFBC相关病变的潜在治疗药物。